Interaction between cyclin D1 and steroid receptor co-activators and uses thereof in assays

ABSTRACT

The present invention relates to the finding that cyclin D1 interacts in a ligand-independent fashion with coactivators of the SRC-1 family. The direct interaction of cyclin D1 enhances estrogen receptor (ER) mediated transcription and provides a novel target for the development of assays for substances which modulate the cell cycle. The invention provides assay methods for the prevention of growth of tumors, for assays for compounds useful in the prevention of tumors and compounds obtainable by such assays.

This is a continuation of U.S. patent application Ser. No. 09/302,305,filed on Apr. 30, 1999 now U.S. Pat. No. 6,350,572, is a continuation ofPCT Application No. PCT/GB99/00440, filed 12 Feb. 1999; which in turnclaims priority from Great Britain Patent Application No. GB 9803035.6,filed 12 Feb. 1998, and Great Britain Patent Application No. GB9818243.9, filed 20 Aug. 1998.

The present invention relates to methods for preventing the growth oftumours, and for assays for compounds useful in the prevention oftumours.

BACKGROUND TO THE INVENTION

The cyclins are a class of polypeptides which are involved in thecontrol of the cell cycle. Three closely related human D-type cyclinshave been identified, all of which interact with and activate cyclindependent kinases (CDK) 4 and 6, although they have specialized functionin distinct cell types. The cyclin D1 gene has been found to beoverexpressed and/or deregulated by clonal chromosome rearrangements orby amplification in B cell lymphoma, in parathyroid adenoma, and inbreast and squamous cell cancer. It has also recently been shown thatcyclin D1 deficient mice have a defect in estrogen-mediatedproliferation of breast epithelium during pregnancy (Sicinski et al,1995, Cell 82; 621-630; Fantl et al, 1995, Genes & Development 9;2364-2372).

Cyclin D1 is induced in response to mitogenic stimulation of quiescentcells and acts as an activator of CDK4 and CDK6. These cyclin D1/CDKcomplexes are key regulators of progression through the GI phase of thecell cycle and are involved in functional inactivation of theretinoblastoma family proteins (reviewed in (Beijersbergen and Bernards,1996)). Cyclin D1 is amplified or over-expressed in a number of humanmalignancies, the most prominent being breast cancer, in which up to 50%of all cases have elevated levels of cyclin D1 (Buckley et al., 1993;Schuuring et al., 1992; van Diest et al., 1997). The relevance of cyclinD1 over-expression is underscored by the finding that tissue-specifictransgenic expression of cyclin D1 in mice results in mammaryhyperplasia and adenocarcinoma (Wang et al., 1994). Furthermore, cyclinD1 knockout mice show a marked defect in breast epithelium developmentduring pregnancy and cyclin D1 reduces mitogen requirement of breastcancer cell lines (Fantl et al., 1995; Musgrove et al., 1994; Sicinskiet al., 1995; Zwijsen et al., 1996). Cyclin D1 is over-expressedpreferentially in ER-positive breast cancers, suggesting that cyclin D1derives (part of) its oncogenic activity in breast cancer by acting onER (Gillett et al., 1996; van Diest et al., 1997). We and others haverecently made a connection between ER and cyclin D1 by showing thatcyclin D1 can interact directly with the ligand binding domain of ER andcan stimulate ER transactivation in a ligand-independent andCDK-independent fashion (Neuman et al., 1997; Zwijsen et al., 1997).

WO97/40378 also discloses that cyclin D1 interacts with the estrogenreceptor (ER). Transcription is increased by formation of cyclin D1-ERcomplex which binds to the estrogen response element (ERE) foundupstream of estrogen responsive genes. This finding provides a targetfor the control of cell proliferation, particularly in those cells whichgrow in response to stimulation by estrogen, e.g. breast tumour cells.

Several lines of evidence suggest that efficient transactivationrequires additional positively acting factors termed coactivators (Pughand Tjian, 1990). Several candidate steroid receptor coactivators (SRCs)have been identified. The first coactivator identified based on itsability to interact with the progesterone receptor was SRC-1 (Onate etal., 1995; Yao et al., 1996). This protein is the founding member of afamily of related SRCs that include TIF-2/GRIP-1 (Hong et al., 1997;Voegel et al., 1996) and AIB-1/ACTR/RAC-3/p/CIP (Anzick et al., 1997;Chen et al., 1997; Li et al., 1997; Torchia et al., 1997). Severalfunctional domains are highly conserved in all members of this family.For instance, the N-terminal regions contain basic helix-loop-helix(bHLH) and per-ARNT-Sim (PAS) domains. Both motifs are thought to beinvolved in protein-protein interactions and DNA-protein interactions(Yao et al., 1996). Interestingly, the bHLH-PAS domain is dispensablefor SRC-1 activity, including receptor interaction and receptoractivation (Onate et al., 1995; Yao et al., 1996). In addition, all SRCscontain multiple LXXLL (SEQ ID NO:6)motifs (L is leucine; X is any aminoacid) in the central region of the protein. These motifs were recentlyshown to be involved in nuclear receptor interaction (Heery et al.,1997; Le Douarin et al., 1996; Torchia et al., 1997). Besides ER, SRC-1also interacts with another coactivator of steroid receptors, CDP/p300,and both types of coactivators act synergistically to enhance ERtransactivation (Chakravarti et al., 1996; Chen et al., 1997; Hansteinet al., 1996; Kamei et al., 1996; Smith et al., 1996; Yao et al., 1996).Both coactivators of the SRC-1 family and the p300/CBP family haveintrinsic histone acetyl transferase (HAT) activity which is widelybelieved to be involved in chromatin remodeling during transcriptionalactivation (Jenster et al., 1997; Ogryzko et al., 1996; Spencer et al.,1997).

DISCLOSURE OF THE INVENTION

We have continued to investigate the mechanism of ER activation bycyclin D1 and found that surprisingly, there is also a directinteraction between SRC-1 and cyclin D1. This interaction appears to bemediated primarily through the LXXLL motifs of SRC-1. We have also foundevidence that other SRCs, particlarly AIB-1.and TIF-2 (which also haveLXXLL motifs) bind to cyclin D1 in a similar manner. The directinteraction of cyclin D1 with SRCs enhances ER mediated transcription.The interaction provides a novel target for the development of assaysfor substances which modulate the cell cycle, particularly in cellswhich grow in response to stimulation by estrogen, e.g. breast tumourcells.

Thus the present invention is useful for assaying for potentialmodulators of the growth of estrogen responsive tumour cells,particularly those in which cyclin D1 is found at elevated levels.Elevated levels of cyclin D1 may occur for a variety of reasons, e.g.over-expression of a single cyclin D1 gene or by gene amplification.

Thus in a first aspect the present invention provides an assay for amodulator of estrogen responsive tumour cells which comprises:

-   a) bringing into contact a cyclin D1, an SRC and a putative    modulator compound under conditions where the cyclin D1 and the SRC,    in the absence of modulator, are capable of forming a complex; and-   b) measuring the degree of inhibition of complex formation caused by    said modulator compound.

The present invention further provides an assay for a modulator ofestrogen responsive tumour cells which comprises:

-   a) bringing into contact a cyclin D1, an SRC, an estrogen receptor    and a putative modulator compound under conditions where the cyclin,    the SRC and the estrogen receptor, in the absence of modulator, are    capable of forming a complex which is capable of binding to an    estrogen response element;-   b) providing an estrogen response element to which the complex is    capable of binding and transcriptionally activating; and-   c) measuring the degree of inhibition of binding or transcriptional    activation caused by said modulator compound.

In a further aspect, the invention provides compounds obtainable by suchan assay, for example peptide compounds based on the portions of cyclinD1, an SRC, or the estrogen receptor which interact with each other.

The assay of the invention is optionally performed in the presence of anestrogen which is capable of binding to the estrogen receptor.

DETAILED DESCRIPTION OF THE INVENTION

Cyclin D1

The cyclin D1 may be any suitable mammalian cyclin D1, particularlyhuman cyclin D1. Human D1 cyclin has been cloned and sources of the genecan be readily identified by those of skill in the art. See for example,Xiong et al, 1991, Cell 65; 691-699 and Xiong et al, 1992, Genomics 13;575-84. Murine D1 cyclin has also been cloned. Other mammalian cyclinscan be obtained using routine cloning methods analogous to thosedescribed in the aforementioned references.

Although wild-type cyclin D1 is preferred mutants and variants of D1which still retain the ability to interact directly with the estrogenreceptor may also be used. Examples of cyclin D1 mutants are well knownin the art and two particular mutants are illustrated in WO97/40378. Aparticularly preferred mutant is a mutant which carries a mutation inthe cyclin box, such as the cyclin D1-KE mutant. This mutation rendersthe D1 unable to bind CDKs.

Mutants and other variants will generally be based on wild-typemammalian cyclin D1s and have a degree of amino acid identity which isdesirably at least 70%, preferably at least 80%, 90%, 95% or even 98%homologous to a wild type mammalian cyclin.

It is not necessary to use the entire cyclin D1 proteins (includingtheir mutants and other variants) for assays of the invention. Fragmentsof the cyclin may be used provided such fragments retain the ability tointeract with the target domain of the SRC and desirably also theestrogen receptor responsible for the cyclin interaction. Fragments ofcyclin D1 may be generated in any suitable way known to those of skillin the art. Suitable ways include, but are not limited to, recombinantexpression of a fragment of the DNA encoding the cyclin. Such fragmentsmay be generated by taking DNA encoding the cyclin, identifying suitablerestriction enzyme recognition sites either side of the portion to beexpressed, and cutting out said portion from the DNA. The portion maythen be operably linked to a suitable promoter in a standardcommercially available expression system. Another recombinant approachis to amplify the relevant portion of the DNA with suitable PCR primers.Small fragments of the cyclin (up to about 20 or 30 amino acids) mayalso be generated using peptide synthesis methods which are well knownin the art. Generally fragments will be at least 40, preferably at least50, 60, 70, 80 or 100 amino acids in size.

Particularly preferred fragments include those which are based upon theC-terminal region of cyclin D1, said region including the C-terminalmotif LLXXXL (SEQ ID NO.7), which in human cyclin D1 is represented bythe sequence LLESSL (SEQ ID NO:8) at residues 254-259. These fragmentswill be able to interact with the SRC and desirably also the ER.Desirably the fragments comprise the C-terminal region of cyclin D1.

The ability of suitable fragments to bind to the SRC (or fragmentthereof) may be tested using routine procedures such as those describedin the accompanying examples relating to intact cyclin D1. Referenceherein to cyclin D1 includes the above mentioned mutants and fragmentswhich are functionally able to bind the SRC unless the context isexplicitly to the contrary.

SRC Protein

The SRC protein may be any human or other mammalian protein, or fragmentthereof which has the ability to bind to a steroid receptor and cyclinD1, and enhance the receptor's transcriptional activity. A number of SRCproteins have been cloned. These include SRC-1, the sequence of which isshown in Onate et al, ibid, 1995; TIF-2, the sequence of which is shownin Voegel et al, ibid, 1996, and AIB-1 (also called ACTR), the sequenceof which is shown in Chen et al, ibid, 1997. These are all human SRCproteins. The sequence of the murine homologue of TIF-2 is disclosed inHong et al, ibid, 1997 (where it is called GRIP-1), and the sequence ofthe murine homologue of AIB-1 is disclosed in Torchia et al, ibid, 1997(where it is called P/CIP).

There are a number of common structural features found in the SRCproteins identified to date. These include an N-terminal region of about300 amino acids comprising the bHLH and PAS regions mentioned above. Acomparision of these domains is shown in Chen et al, ibid. The AIB-1bHLH/PAS domain shares about 65% homology (identity) with thecorresponding TIF-2 domain and about 58% homology (identity) with thecorresponding SRC-1 domain.

The central portion of the proteins (from about amino acids 570 to 780of SRC-1) comprises a receptor interaction domain (RID) which alsoappears to be primarily responsible for the physical interaction withcyclin D1. The AIB-1 RID has 42% and 27% amino acid identity with theTIF-2 and SRC-1 RIDs respectively.: The RIDs comprise three motifs whichshare the common primary sequece LXXLL (SEQ ID NO:6).

The SRCs also have, C-terminal to the central portion, a histone acetyltransferase domain.

Thus an SRC protein in its broadest sense is one which has, in N- toC-terminal order, a bHLH/PAS domain of about 300 amino acids with atleast 50% homology to the corresponding domain of SRC-1, AIB-1 or TIF-2;a centrally located RID of about 210 amino acids with one or more, suchas three, LXXLL (SEQ ID NO:6) motifs; and a domain which has HATactivity.

Variants of the above SRCs may be used, such as synthetic variants whichhave at least 50% amino acid identity to a naturally occurring SRC(particularly a human SRC), preferably at least 60%, 70%, 80%, 90%, 95%or 98% identity. The assay preferably uses the same mammalian source SRCas the cyclin D1. A preferred subset of variants are those which retainone or more of the motifs LXXLL (SEQ ID NO:6) of the central portion,and preferably retains three as found in the SRCs mentioned above. Thevariants may also be manipulated to provide more than three, forexample, four, five or six, LXXLL (SEQ ID NO:6) motifs. These additionalmotifs will preferably be within the RID domain, but may also beintroduced in other portions of an SRC protein. The motifs may beintroduced by insertion of additional amino acids of by substitution ofamino acids within the SRC.

Fragments of the SRC protein and its variants may be used, provided thatsaid fragments retain the ability to interact with a wild-type cyclinD1, preferably wild-type human cyclin D1. Such fragments are desirablyat least 50, preferably at least 75, 100, 200, 250 or 400 amino acids insize. Desirably such fragments include one or more of the (such as allthree of the centrally located) LXXLL (SEQ ID NO:6) motifs.

Estrocren Receptor

The estrogen receptor used in the assay may be obtained from the samemammalian source as the cyclin D1. The human estrogen receptor ispreferred. This is a 66 kd protein which functions as hormone-activatedtranscription factor, the sequence of which is published in the art andis generally available. Receptor activation is thought to be aconsequence of ligand-induced conformational changes in the structure ofthe receptor. The complex of estrogen with its receptor binds tospecific DNA sequences including, with high affinity, to a well-defined13-bp palindromic sequence—the estrogen response element (ERE). The EREis usually located upstream of an estrogen-responsive gene. Estrogenresponsive genes include progesterone receptor and PS-2. Transcriptionalactivation of these genes is involved in estrogenresponsive tumourgrowth.

There is also a second estrogen receptor, ER beta. This is disclosed inKuiper et al, 1996. The ERβ may also be used, and is included hereinwhen reference is made to the ER.

Fragments and variants of the estrogen receptor which retain the abilityto interact with the cyclin D1 may also be used, and such fragments andmutants may be obtained by methods analogous to those described above inrelation to cyclin fragments.

Variants of ER include a number of variants which have been found to beassociated with breast cancer. Some of these are associated with breastcancers which are resistant to anti-estrogen therapies, particularlytamoxifen therapies. Variants are disclosed in, for example, McGuire etal, 1991; Fuqua et al, 1993; and Miksicek et al, 1995.

Mutants and other variants of ER will generally be based on wild-typemammalian ERs and have a degree of amino acid identity which isdesirably at least 70%, preferably at least 80%, 90%, 95% or even 98%homologous to a wild type mammalian ER.

Particluarly preferred fragments (including fragments of theabove-described variants) include those which retain the E/F regionscomprising amino acids 292-595 or a portion thereof such as 340-595 ofthe ER. Smaller fragments may also be used, e.g. those starting ataround 292, 300, 340, 360 or 380 and ending at the C-terminus (595) or atruncation thereof, e.g. 590, 580, 550 or 500. Suitable fragments may bedetermined by routine experimentation. Reference herein to the estrogenreceptor includes fragments and mutants which retain the ability tointeract with cyclin D1 unless the context is explicitly to thecontrary. In this context, “interact” includes binding to ER, theminimum requirement for an in vitro assay. For in vitro or in vivoassays which rely on the D1-SRC-ER complex binding to an ERE andactivating transcription, the necessary interaction must provide this.

In an alternative format the ER may be added in the form of two partialproteins, for example expressed from two partial ER expression vectors.The first protein encodes the N-terminal region of ER (containing AF-1and the DNA binding region), the second the C-terminal part (containingAF-2 and the hormone binding region). The hormone binding region isoptionally linked to an activator protein such as the GAL4 activationdomain or the VP16 activation domain. As illustrated in the accompanyingexamples, ERE-dependent transcription is stimulated by both cyclin D1and an SRC protein, and the presence of both components synergisticallyenhances the activation. Thus putative modulators may be used and thedecrease in the synergistic enhancement of activation may be observed inassays of the present invention.

Estrogens

Although we previously have found that the estrogen receptor isactivated by cyclin D1 alone to provide ERE-responsive genetranscription we have found that transcription is enhancedsynergistically in the presence of an estrogen. It is thus a preferredaspect of the assay that estrogen is also brought into contact with thecyclin D1, SRC, estrogen receptor and putative inhibitor compound when atranscriptional acitivation assay of the invention is performed. Theestrogen may be any natural or synthetic estrogen capable of binding toand activating the estrogen receptor. Examples of estrogens include17β-estradiol.

Assay Formats

One assay format which is widely used in the art to study the ainteraction of two proteins is a two-hybrid assay. This assay may beadapted for use in the present invention. A two-hybrid assay comprisesthe expression in a host cell of the the two proteins, one being afusion protein comprising a DNA binding domain (DBD), such as the yeastGAL4 binding domain, and the other being a fusion protein comprising anactivation domain, such as that from GAL4 or VP16. In such a case thehost cell (which again may be bacterial, yeast, insect or mammalian,particularly yeast or mammalian) will carry a reporter gene constructwith a promoter comprising a DNA binding elements compatible with theDBD. The reporter gene may be a reporter gene such as chloramphenicalacetyl transferase, luciferase, green fluorescent protein (GFP) andβ-galactosidase, with luciferase being particularly preferred.

Two-hybrid assays may be in accordance with those disclosed by Fieldsand Song, 1989, Nature 340; 245-246. In such an assay the DNA bindingdomain (DBD) and the transcriptional activation domain (TAD) of theyeast GAL4 transcription factor are fused to the first and secondmolecules respectively whose interaction is to be investigated. Afunctional GAL4 transcription factor is restored only when two moleculesof interest interact. Thus, interaction of the molecules may be measuredby the use of a reporter gene operably linked to a GAL4 DNA binding sitewhich is capable of activating transcription of said reporter gene.

Thus two hybrid assays may be performed in the presence of a potentialmodulator compound and the effect of the modulator will be reflected inthe change in transcription level of the reporter gene constructcompared to the transcription level in the absence of a modulator.

Host cells in which the two-hybrid assay may be conducted includemammalian, insect and yeast cells, with yeast cells (such as S.cerivissiae and S. pombe) being particularly preferred.

In the case of the present invention, a two-hybrid assay will beconducted by fusing the cyclin D1 protein being used to a DNA-bindingdomain and the SRC protein to the activation domain.

Another assay format measures directly, in vivo or in vitro theinteraction between cyclin D1 and the SRC by labelling one of theseproteins with a detectable label and bringing it into contact with theother protein which has been optionally immobilised on a solid support,either prior to or after proteins have been brought into contact witheach other. Suitable detectable labels include ³⁵S-methionine which maybe incorporated into recombinantly produced proteins, and tags such asan HA tag, GST or histidine. The recombinantly produced protein may alsobe expressed as a fusion protein containing an epitope which can belabelled with an antibody. Alternatively, an antibody against the cyclinand/or SRC can be obtained using conventional methodology.

The protein which is optionally immobilized on a solid support may beimmobilized using an antibody against that protein bound to a solidsupport or via other technologies which are known per se. In theExamples which follow a preferred in vitro interaction is illustratedwhich utilises a fusion protein of SRC-1 fused toglutathione-S-transferase (GST). This may be immobilized on glutathioneagarose beads. In an in vitro assay format of the type described abovethe putative inhibitor compound can be assayed by determining itsability to diminish the amount of labelled cyclin D1 which binds to theimmobilized GST-SRC-1. This may be determined by fractionating theglutathione-agarose beads by SDS-polyacrylamide gel electrophoresis.Alternatively, the beads may be rinsed to remove unbound protein and theamount of protein which has bound can be determined by counting theamount of label present, in, for example, a suitable scintillationcounter.

Another assay format is dissociation enhanced lanthanide fluorescentimmunoassay (DELFIA) (Ogata et al, 1992). This is a solid phase basedsystem for measuring the interaction of two macromolecules. Typicallyone molecule (either SRC1 or cyclin D1) is immobilised to the surface ofa multi well plate and the other molecule is added in solution to this.Detection of the bound partner is achieved by using a label consistingof a chelate of a rare earth metal. This label can be directly attachedto the interacting molecule or may be introduced to the complex via anantibody to the molecule or to the molecules epitope tag. Alternatively,the molecule may be attached to biotin and a streptavidin-rare earthchelate used as the label. The rare earth used in the label may beeuropium, samarium, terbium or dysprosium. After washing to removeunbound label, a detergent containing low pH buffer is added todissociate the rare earth metal from the chelate. The highly fluorescentmetal ions are then quantitated by time resolved fluorimetry. A numberof labelled reagents are commercially available for this technique,including streptavidin, antibodies against glutathione-S-transferase andagainst hexahistidine.

In an alternative mode, the one of the cyclin D1 and the SRC may belabelled with a fluorescent donor moiety and the other labelled with anacceptor which is capable of reducing the emission from the donor. Thisallows an assay according to the invention to be conducted byfluorescence resonance energy transfer (FRET). In this mode, thefluorescence signal of the donor will be altered when the cyclin D1 andan SRC interact. The presence of a candidate modulator compound whichmodulates the interaction will increase or decrease the amount ofunaltered fluorescence signal of the donor.

FRET is a technique known per se in the art and thus the precise donorand acceptor molecules and the means by which they are linked to thecyclin D1 and an SRC may be accomplished by reference to the literature.

Suitable fluorescent donor moieties are those capable of transferringfluorogenic energy to another fluorogenicmolecule or part of a compoundand include, but are not limited to, coumarins and related dyes such asfluoresceins, rhodols and rhodamines, resorufins, cyanine dyes, bimanes,acridines, isoindoles, dansyl dyes, aminophthalic hydrazines such asluminol and isoluminol derivatives, aminophthalimides,aminonaphthalimides, aminobenzofurans, aminoquinolines,dicyanohydroquinones, and europium and terbium complexes and relatedcompounds.

Suitable acceptors include, but are not limited to, coumarins andrelated fluorophores, xanthenes such as fluoresceins, rhodols andrhodamines, resorufins, cyanines, difluoroboradiazaindacenes, andphthalocyanines.

A preferred donor is fluorescein and preferred acceptors includerhodamine and carbocyanine. The isothiocyanate derivatives of thesefluorescein and rhodamine, available from Aldrich Chemical Company Ltd,Gillingham, Dorset, UK, may be used to label the cyclin D1 and ER. Forattachment of carbocyanine, see for example Guo et al, J. Biol. Chem.,270; 27562-8, 1995.

The assays described above may also utilise the requirement of SRC andER for an estrogen if binding of ER to the estrogen response element(ERE) is to occur. We have found that the binding of ER to the ERE isindependent of estrogen when either cyclin D1 is present, or both cyclinD1 and an SRC are present. The binding of ER to the ERE provides thebasis for a binding assay. For example, a detectably labelled ERE may bebrought into contact with ER, cyclin D1 and an SRC under conditionswhere, in the absence of an inhibitor, a complex of the three componentsis detectable on the ERE. The ERE is preferably labelled with a labelwhich allows physical recovery of the ERE, for example with aparamagnetic bead or a protein which may be reversibly or irreversiblyattached to a solid support such as a chromatography column.

Once the ERE is recovered, the proteins still associated with it may berecovered and the presence or absence of specific components of thecomplex determined. Protein components may conveniently be examined byWestern blotting. Alternatively, immunological means may be used. Inanother alternative, the different protein components may be labelledwith different fluorogenic or radioactive label, which may be detectedwith little or no separation of the recovered ERE-protein complex.

Such assays may be used to determine whether, in the presence of aputative modulator compound, the SRC remains bound to cyclin D1 in theternary complex.

The assay of the invention may also take the form of an in vivo assaybased on the ability of the complex of the ER, cyclin D1 and the SRC toactivate estrogen responsive transcription. The term “in vivo” includescell lines and the like, and excludes whole humans. The in vivo assaymay be performed in an estrogen responsive cell line which expresses theestrogen receptor or in ER-negative cell lines in which the ER isexpressed from a vector introduced into the cell. The cell line may bein tissue culture or may be a cell line xenograft in a non-human animalsubject.

Where a cell line expressing the estrogen receptor is used a reportergene construct comprising an ERE operably linked to a reporter gene maybe introduced into the cell together with vector(s) for the expressionof a cyclin D1 and an SRC. These proteins may be expressed from a singlevector or from two separate vectors. The vector(s) may utilize anysuitable promoter, such as described herein. Two or more EREs (forexample 3, 4 or 5) may be present in the receptor construct and this mayenhance sensitivity of the assay. The reporter gene may be any suitablereporter gene used in the art. Such reporter genes includeschloramphenicol acetyl transferase (CAT), β-galactosidase, luciferase orGFP. The cyclin D1 expression vector will comprise DNA encoding cyclinoperably linked to a promoter capable of expressing the gene in the hostcell. Suitable promoters include viral promoters such as a CMV or SV40promoter.

The cell lines used in assays of the invention may be used to achievetransient expression of the cyclin although in a further aspect of theinvention cells which are stably transfected with constructs whichexpress a D1 and SRC and, where required, the ER may also be generated.Means to generated stably transformed cell lines are well known in theart and such means may be used here.

Suitable cell lines include breast cancer cell lines which are widelyavailable in the art. The Examples which follow utilise the T47D breastcancer cell line although other suitable Examples include MCF-7 and MDA.Such assays may also be performed in cell free systems such as areticulocyte cell free system.

Where the cell line does not express ER, a construct capable ofexpressing this protein may also be introduced into the cell operablylinked to a suitable promoter.

The precise format of the assays of the invention may be varied by thoseof skill in the art using routine skill and knowledge. In the in vitroassays of the invention, the amount of cyclin D1, SRC and, whererequired, estrogen receptor may be varied depending upon the scale ofthe assay. In general, the person of skill in the art will selectrelatively equimolar amounts of the two components, say from 1:10 to100:1, preferably from 1:1 to 10:1, molar ratio of cyclin D1 to SRC.However there may be particular assay formats which can be practicedoutside this range.

In the in vivo assays of the invention, it will be desirable to achievesufficient expression of cyclin D1 to recruit sufficient SRC to acomplex with ER that the effect of a putative modulator compound may bemeasured. Where the cell does not express ER, sufficient expression ofthis will also be required. The level of expression of cyclin D1 and SRC(and where necessary ER) may be varied within fairly wide limits, sothat the intracellular levels of the two may vary by a wide ratio, forexample from 1:10 to 1000:1, preferably 1:1 to 100:1, molar ratio ofcyclin D1 to SRC.

While not wishing to be bound by any one theory, the interaction of SRCto both ER and cyclin D1 through similar regions and motifs of SRCsuggests that SRC interaction with these components may occur throughdimerization of SRC. It may therefore be desirable when performing theinvention, particularly in the presence an ER, to ensure sufficientexcess of SRC to allow for dimerization to occur. In addition, thedimerization of SRC gives rise to a further novel target for potentialmodulator compouds, wherein such assays may be conducted as describedherein for SRC-cyclin D1 assays, with the D1 component being replace bya second SRC, either identical to or different from the first SRC.

The amount of putative modulator compound which may be added to an assayof the invention will normally be determined by trial and errordepending upon the type of compound used. Typically, from about 0.01 to100 nM concentrations of putative modulator compound may be used, forexample from 0.1 to 10 nM. Modulator compounds may be those which eitheragonise or antagonise the interaction. Antagonists (inhibitors) of theinteraction are particularly desirable.

Modulator compounds which may be used may be natural or syntheticchemical compounds used in drug screening programmes. Extracts of plantswhich contain several characterised or uncharacterised components mayalso be used. Modulators which are putative inhibitor compounds can bederived from the cyclin D1 and SRC protein sequences. Peptide fragmentsof from 5 to 40 amino acids, for example from 6 to 10 amino acids fromthe region of cyclin D1 and SRC which are responsible for theinteraction between these proteins may be tested for their ability todisrupt this interaction. Antibodies directed to the site of interactionin either protein form a further class of putative inhibitor compounds.Candidate inhibitor antibodies may be characterised and their bindingregions determined to provide single chain antibodies and fragmentsthereof which are responsible for disrupting the interaction betweencyclin D1 and SRC.

A particular class of peptide compounds will be those based upon “LXXLL”(SEQ ID NO:6) peptides shown in the accompanying examples. Such peptidesare preferably form 5 to 20 amino acids in size, and are of thestructure Z¹-LXXLL-Z² (SEQ ID NO:9) where Z¹ is an N-terminal or asequence of from 1 to 8 amino acids, preferably a sequence foundimmediately N-terminal to a naturally occuring LXXLL (SEQ ID NO:6) motifin an SRC, and Z² is a terminal or sequence of from 1 to 8 amino acids,preferably a sequence found immediately C-terminal to a naturallyoccuring LXXLL (SEQ ID NO:6) motif in an SRC or other nuclear receptorinteracting protein such as p300 or CBP. Z¹ and Z² may be from the sameor different SRC and where from the same, from the same or differentmotif.

Particular peptides according to this aspect of the invention includethose shown in Table 1 below (the peptides are shown in conventional N-to C-terminal order using the standard 1-letter code:

TABLE 1 A A S K H K Q L S E L L R S G SEQ ID NO:1         S H K L V Q LL T T T A E Q SEQ ID NO:2     E R H K I L H R L L Q E G S SEQ ID NO:3    K D H Q L L R Y L L D K D E SEQ ID NO:4 P Q A Q Q K S L Q Q L L TSEQ ID NO:5

In a preferred aspect of the invention, Z¹ comprises from 3 to 7 aminoacids and contains at least one, such as 1, 2 or 3, residue selectedfrom the group of K and H. In this embodiment of the invention, it ispreferred that-the amino acid at position −1, −2 or −3, preferably −2,with respect to the first L of the LXXLL (SEQ ID NO:6) motif is K or H.

Although the second and third amino acids of the LXXLL motif may vary,it is preferred that at least one of said amino acids is charged.

We have also found that peptides of this aspect of the invention areactive even where Z² comprises a single amino acid. Thus, in a preferredaspect of the invention, z² may be from 1 to 4 amino acids in length.

The abovementioned preferred features may be present separately or inany combination in peptides of the invention.

Peptides comprising an LXXLL (SEQ ID NO:6) motif may comprise multimersof this motif, either in the form of one or more direct repeats, ofseparated by one or more amino acids. The peptides may also be branchedpeptide structures, referred to in the art as dendrimers.

Peptide antagonists of the interaction of cyclin D1 with an SRC may belinked, at the C- or N-terminal, to a member of the class of sequenceswhich are membrane translocation sequences capable of directing apolypeptide through the membrane of a eukaryotic cell. Example of suchpolypeptides include the HSV-1 VP22 protein (Elliot et al, 1997), theHIV Tat protein (for example residues 1-72 or 37-72, Fawell et al, 1994)or a sequence that is derived from the Drosophila melanogasterantennapedia protein. The latter is a peptide containing 16 amino acidresidues taken from the third helix of the antennapedia homeodomainprotein which translocates across biological membranes (Derossi et al,1994). This translocation peptide has the sequence:Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys (SEQ IDNO:6). The peptide is preferably joined to the N-terminus ofpolypeptides of the invention which antagonize the interaction of cyclinD1 with an SRC.

Other candidate inhibitor compounds may be based on modelling the3-dimensional structure of cyclin D1 and SRC and using rational drugdesign to provide potential inhibitor compounds with particularmolecular shape, size and charge characteristics.

Candidate modulator compounds obtained according to the method of theinvention may be prepared as a pharmaceutical preparation. Suchpreparations will comprise the compound together with suitable carriers,diluents and excipients. Such formulations form a further aspect of thepresent invention. Formulations of inhibitor compounds in particular maybe used in methods of treatment of proliferative diseases, particularlythose involving cells which grow in response to stimulation by estrogen,for example breast cancer.

Candidate inhibitor compounds may also be used in combination with anyother anti-proliferative compounds used to treat hyperproliferativediseases such as cancers. In such a case, the assay of the invention,when conducted in vivo, need not measure the degree of inhibition ofbinding or transcriptional activation caused by the inhibitor compoundbeing tested. Instead the effect on cell growth or proliferation may bemeasured. It may be that such a modified assay is run in parallel orsubsequent to the main assay of the invention in order to confirm thatany effect on cell growth or proliferation is as a result of theinhibition of binding or transcriptional activation caused by saidinhibitor compound and not merely a general toxic effect.

A preferred class of other anti-proliferative compounds areanti-estrogens such as tamoxifens, eg. 4-hydroxytamoxifen or pureanti-estrogens such asN-(n-butyl)-11-[3,17beta-dihydroxy-estra-1,3,5(10)-trien-7alpha-yl]N-methylundecanamide,known as ICI 164,384. Such anti-estrogen compounds may be included inthe assay in order to determine inhibitor compounds which may actadditively or synergistically with anti-estrogens. This will facilitatethe provision of combination therapy against estrogen-responsivetumours.

Estrogen responsive tumours are primarily breast tumours although othertumour types which have been found to be estrogen-responsive includeendometrial cancer and ovarian carcinoma.

The interaction between cyclin D1 and the SRC may also be used tomonitor the status and progress of disease states associated withenhanced transcription of ERE regulated genes. For example, antibodiesmay be generated which bind to the cyclin/SRC complex or to one or othercomponent in the allosteric form induced by binding to the other. Suchantibodies may be raised in routine ways, using as immunogen thecyclin/SRC complex which may be stabilized by using proteincross-linking reagents. The antibodies generated by such an immunogenmay be screened against the immunogen and separately against the cyclinand SRC, in order to obtain antibodies which recognise only the proteinswhen complexed. Such antibodies may be packaged in kits with othersuitable reagents for immunodiagnosis and used in the clinic to monitordisease states.

The following Examples illustrate the invention.

Legends to Figures.

FIG. 1. Mapping of the region of cyclin D1 required for ER-mediatedtransactivation.

-   (A) The effect of cyclin D1 deletion mutants on ER activation in the    presence of ligand. The cyclin D1 derivatives used in this study are    shown in the left panel; right side of Figure represents the    relative capacity of the mutants to potentiate ERE-dependent    transcription. ER-negative COS-7 cells were transfected with    expression vectors for wild type ER (200 ng), cyclin D1 or cyclin D1    mutants (2.5 μg), an internal control §-galactosidase plasmid (0.5    μg) and an ERE-TATA-luciferase reporter (3 μg). The effect on ER    transactivation of wild type cyclin D1 was set to 100%. These    studies were performed in three separate experiments and expressed    as mean values with standard deviation less then Ups. The alignment    of leucine-rich motif of ER with D-type cyclins are shown on the    bottom; the leucine-rich motif in cyclin D1 is indicated as a dark    box. The identifiers for the sequences shown are as follows: Cyclin    D1 (SEQ ID NO:11), Estrogen receptor (SEQ ID NO:12), the Cyclin D2    (SEQ ID NO:13), and the Cyclin D3 (SEQ ID NO:14).-   (B) ER activation by D-type cyclins, cyclin D1 L254/255A point    mutant (D1-LALA) and SRC-1 in the presence of ligand. COS-7 cells    were transfected with D-type cyclin expression vectors, cyclin D1    Leucine-to-Alanine point mutant (D1-LALA) or SRC-1 expression vector    together with wild type ER expression vector, an ERE-TATA-luciferase    reporter plasmid and the internal control β-galactosidase construct.    Data are expressed as relative luciferase activity compared with    basal ERE-luciferase activity of wild type ER and are normalized for    transfection efficiencies.

FIG. 2. Effect of cyclin D1 on helix 12 mutants of ER. The effect ofcyclin D1 on ER mutants was tested in COS-7 cells (A, B) and in U2-OScells (C, D) in the presence of ligand. An ERE-TATA-luciferase reporterconstruct was used in transient transfections together with cyclin D1and ER 1-535 (A, C) or ER L543/544A mutants (B, D). β-Galactosidaseserved as an internal control. The reporter activity was determined bothin the presence (black bars) and in the absence (white bars) of 10 nM17β-estradiol. The relative luciferase activity was calculated bynormalizing to the b-galactosidase activity. The relative reporteractivity of wild type ER in the presence of ligand was used as areference and set at 100%. In the absence of transfected ER plasmid,cyclin D1 did not induce transcriptional activity of the reporter (datanot shown). The transfection were performed in at least five separateexperiments and expressed as average ±SD.

FIG. 3. Role of coactivators in cyclin D1—induced transactivation.

-   (A) Effect of a dominant negative form of SRC-1 (SRC1-DN) on ER    transactivation. SRC1-DN encoding amino acids 1245-1441 of SRC-1 (1,    2.5 and 5 μg) was introduced by transient transfection together with    wild type ER (200 ng) and was tested for its ability to modulate    ERE-dependent transcription.-   (B) Effect of SRCI-DN on SRC-1 and TIF 2 mediated ER    transactivation. SRC-1 (3 μg) or TIF-2 (3 μg) were transfected with    SRC1-DN (3 μg) and tested for ER transactivation.-   (C) Effect of SRC1-DN on cyclin D1-induced transactivation of ER    1-535 mutant. Cyclin D1 was cotransfected with SRC1-DN (1, 2.5, 5    μg) and tested for its effect on an ER harboring a deletion of    coactivator binding site in helix 12 (ER 1-535).-   (D) Effect of SRC1-DN on cyclin D1-induced transactivation of ER    L543/544A mutant. Cyclin D1 and SRC1-DN (1, 2.5, 5 gg) were    transfected and tested for the activity of ER helix 12 point mutant    (ER L543/544A). The transient transfections (A-C) were performed in    COS-7 cells, which were maintained in DMEM with 10 nM ligand. The    relative activity was calculated by normalizing to the internal    control and was divided by luciferase activity of ER (mutant) in the    presence of ligand. The transfection for each set of conditions were    done at least in four independent experiments and expressed as    average ±SD.

FIG. 4A. Structure of GST-SRC1 derivatives used to show that amino acids568 to 782 of SRC-1 are required for cyclin D1 binding.

FIG. 4B. Sequences of peptides P1(SEQ ID NO:15), P2(SEQ ID No:16),P3(SEQ. ID No:17), P4(SEQ ID NO:18), and P4M (SEQ ID NO:19).

FIG. 5. Cyclin D1 mediates ligand-independent recruitment of SRC-1 toER.

-   (A) Ligand-independent in vitro binding of SRC-1, ER and cyclin D1.    The purified proteins GST-SRC1, His-tagged cyclin D1 and    baculovirus-produced ER were tested for in vitro binding-in a    GST-pull down assay. GST protein served as negative control. Cyclin    D1 and ER were incubated with GST-SRC1 in the presence or absence of    1 μM 17β-estradiol and binding was detected by Western blot analysis    using anti-cyclin D1 and anti-ER monoclonal antibodies. Lane 1    represents 10%of the input for cyclin D1 and 20% of input of ER    proteins.-   (B) Cyclin D1 and SRC-1 can interact with DNA-bound ER.    Oligonucleotide containing ER binding sequence was biotin 5′-end    labeled and bound to paramagnetic particles coated with    streptavidin. Purified GST-SRCl, baculovirus produced ER and    His-tagged cyclin D1 proteins were tested for DNA binding using    these ERE-containing beads and analyzed by Western blotting using    antibodies directed against GST, ER and cyclin D1, respectively.

FIG. 6. Role of cyclin D1 in ER transactivation in breast cancer cells.

The ER-containing T47D and MCF-7 breast cancer cells were maintained in17β-estradiol enriched medium with 10%fetal calf serum aftercotransfection with a cyclin D1 L254/255A mutant expression vector andan ERE-reporter gene for testing its effect on ER activation (leftpanel). ER transactivation in the absence of co-expression of the cyclinD1 mutant was set at 100%. The right panel shows the expression levelsof endogenous cyclin D1 in both breast tumor cell lines.

FIG. 7. Model for cyclin D1-mediated ER transactivation. In the absenceof ligand, ER is unable to interact with SRCs directly as itsleucine-rich coactivator interaction motif is sterically unavailable forinteraction. Ligand-independent binding of cyclin D1 to ER provides asingle leucine-rich interaction motif for SRCs which is present in thecarboxyl terminus of cyclin D1. This results in partial activation of ER(left panel). Subsequent ligand binding of ER induces a conformationalchange in ER that also exposes the leucine-rich motif in AF-2 of ER forSRC interaction, allowing higher affinity binding of SRCs to theliganded D1/ER complex (right panel). The observed synergism betweenestradiol and cyclin D1 in ER activation results from their cooperativerecruitment of SRCs to the D1/ER complex. The protein interaction motifsare shown in italics. The identifiers for the depicted motifs are: LXXLL(SEQ ID NO:6) and LLXXXL (SEQ ID NO:20).

The invention is illustrated by the following examples.

The estrogen receptor (ER) belongs to the steroid/nuclear receptorfamily of ligand-regulated transcription factors. Members of thissuperfamily display a modular structure with six distinct functionalregions (termed A-F), which includes domains for DNA binding, ligandbinding and transcriptional activation. Like other members of thenuclear hormone receptor superfamily, ER harbors two transcriptionalactivation functions (AFs) that act synergistically in transactivation(Kumar et al., 1987; Tzukerman et al., 1994). Transcriptional activationis mediated by means of the autonomous activation function (AF-1) in theN-terminal A/B domain and the ligand-dependent activation function(AF-2) in the C-terminal hormone binding domain (Evans, 1988; Kumar andChambon, 1988; Beato, 1989). These two regions flank the DNA-bindingdomain of the receptor. Upon ligand binding, ER binds to estrogenresponsive elements, which results in activation of specific ER targetgenes (Beato, 1989).

It is generally thought that nuclear receptors stimulate transcriptionthrough direct binding to several of the basal transcription factors,thereby enhancing the formation of a stable transcription pre-initiationcomplex (Mitchell and Tjian, 1989). This notion is supported by in vitroprotein binding studies which demonstrated that several steroidreceptors interact directly with components of the basal transcriptionalapparatus, including the TATA-box-binding protein TBP (Sadovsky et al.,1995), TFIIB (Ing et al., 1992; Baniahmad et al., 1993) and humanTAF_(II)30 (Jacq et al., 1994). However, as described above, severallines of evidence suggest that efficient transactivation requiresadditional positively acting factors termed coactivators.

Transactivation by steroid/nuclear receptors involves the well-conservedAF-2 domain located in helix 12 of the carboxyl-terminus of thereceptors. It has been demonstrated that helix 12 harbors a leucine-richmotif that constitutes a ligand-regulated binding site for coactivators,like SRC-1 (Danielian et al., 1992; Le Douarin et al., 1995; Voegel etal, 1996; vom Baur et al., 1996). Consequently, transactivation bynuclear receptors is dramatically reduced in receptors that containmutations in helix 12 (Danielian et al., 1992; White et al., 1997). Theleucine-rich motif in helix 12 of nuclear receptors is involved inbinding to the LXXLL (SEQ ID NO:6) motifs of the steroid receptorcoactivators (Le Douarin et al., 1996; Heery et al., 1997).

Recently it was found that, apart from cyclin D1, the steroid receptorcoactivator AIB-1 is also frequently amplified in breast cancer (Anzicket al., 1997). In the present work we describe an unexpectedrelationship between cyclin D1 and steroid receptor coactivators whichplaces cyclin D1 at the center of a complex transcription regulatorynetwork of nuclear hormone receptors and their coactivators. We identifya novel functional domain in cyclin D1 that mediates direct interactionwith several of the steroid receptor coactivators.

ER and Cyclin D1 Share a Coactivator Binding Motif.

To study how cyclin D1 activates ER, cyclin D1 deletion mutants weretested for their effect on ER-transactivation. COS-7 cells weretransfected with cyclin D1 mutants, together with ER and a luciferasereporter gene construct driven by a minimal TATA promoter linked to anestrogen response element (ERE). FIG. 1A shows that an amino-terminaldeletion mutant of cyclin D1 (D1: amino acids 91-295) was still able toactivate ER, whereas two carboxyl-terminal deletion mutants of cyclin D1(D1: amino acids 1-202 and D1: amino acids 1-247) lack ERtransactivation capacity. A cyclin D1 mutant lacking the extremecarboxyl-terminus (D1: amino acids 1-267) partially retained ERactivation. Together, these data indicate that the domain required forER activation is located in the carboxyl-terminal 48 amino acids ofcyclin D1. This part of the protein is not involved in CDK interactionand is poorly conserved among the different cyclins. Alignment ofsequences in this part of cyclin D1 with ER revealed that a motif thatresembles the highly conserved leucine-rich coactivator binding motif inAF-2 of ER is present within the domain of cyclin D1 implicated in ERtransactivation at the amino acid positions 254-259 (FIG. 1A). Thismotif is only partially conserved in cyclin D2 and D3, two cyclins thatare far less active in ER transactivation (Neuman et al., 1997; Zwijsenet al., 1997). To test the relevance of this leucine-rich domain ofcyclin D1 in ER activation, a cyclin D1 mutant was constructed in whichleucines 254 and 255 were mutated to alanines (D1 L254/255A). Thismutation in cyclin D1 is similar to the mutation in ER (ER L543/544A)that interferes with coactivator binding to AF-2 (Danielian et al.,1992). In contrast to wild type cyclin D1, the L254/255A mutant cyclinD1 was virtually unable to activate wild type ER even though this mutantwas equally well expressed and was fully active in other assays (FIG. 1Band see be-low). Cyclins D2 and D3, which lack this leucine-rich motif,behaved similar-to the D1 L254/255A mutant in ER activation (FIG. 1B).These data suggest that cyclin D1 can activate ER through an AF-2-likemotif.

Activation of AF-2 Mutant ERs by Cyclin D1.

It has been demonstrated that AF-2 mutant ERs are unable to activatetranscription, because they cannot recruit steroid receptor coactivators(SRCs) efficiently (Danielian et al., 1992; vom Baur et al., 1996). Todetermine the role of the AF-2 domain of ER in cyclin D1-mediatedtransactivation, a deletion mutant and a point mutant in theligand-regulated carboxyl-terminal activation domain (AF-2) of ER weretested in COS-7 cells. FIG. 2 shows that, as previously reported, theactivity of these AF-2 mutant ERs reflects background levels (Danielianet al., 1992; vom Baur et al., 1996). Surprisingly, co-expression ofcyclin D1 resulted in a significant activation of the ER AF-2 deletionmutant (ER 1-535) to levels that were up to 40% of ligand-activated wildtype receptor (FIG. 2A). Cyclin D1 was also able to induce transcriptionin the absence of ligand, although this increase was less pronounced.Similarly, ER L543/544A, which harbors a mutation in the leucine-richcoactivator binding site in AF-2 (LLXXXL (SEQ ID NO:20) to AAXXXL (SEQID NO:21)), could be activated by cyclin D1 (FIG. 2B). Comparableresults were obtained in ER-negative U2-OS osteosarcoma cells (FIGS. 2C,D), indicating that the effect of cyclin D1 is not cell type-specific.These results indicate that cyclin D1 can mediate activation of ERmutants that are unable to interact with SRCs efficiently.

To ask whether SRCs are involved in the cyclin D1-mediated activation ofthe mutant ERs, a dominant negative mutant of SRC-1 (SRC1-DN, encodingamino acids 1245-1441 of SRC-1) was used. This mutant harbors-a LXXLL(SEQ ID NO:6) motif, which mediates binding to the leucine-richcoactivator binding site in ER (Heery et al., 1997; Le Douarin et al.,1995) but lacks a transactivation domain (Jenster et al., 1997; Spenceret al., 1997). As shown in previous studies (Onate et al., 1995), thisconstruct served as a dominant inhibitor for endogenous SRC-1 functionon wild type ER (FIG. 3A). As expected, SRCI-DN inhibited the ability ofSRC-1 and of the closely related coactivator TIF2 on ER transactivation(FIG. 3B), whereas it was inactive on the non-related E2F-1transcription factor (data not shown) Importantly, SRC1-DN markedlyrepressed the cyclin D1-induced activation of the ER AF-2 mutants (FIGS.3C, D). These data suggest that the cyclin D1-mediated activation ofthese mutant ERs somehow requires SRC activity.

Direct Binding of Cyclin D1 to Steroid Receptor Coactivators.

Since the leucine-rich motifs of nuclear receptors have been shown torecruit SRC family coactivators (Danielian et al., 1992; Heery et al.,1997), we tested whether cyclin D1 is also able to interact with SRCsthrough its leucine-rich motif. COS-7 cells were cotransfected withHA-tagged constructs encoding the nuclear receptor coactivators SRC-1,AIB-1 or p300 (Anzick et al., 1997; Chakravarti et al., 1996; Hansteinet al., 1996; Kamei et al., 1996; Onate et al., 1995) together withcontrol plasmid, plasmids directing the synthesis of wild type cyclin D1or D1 L254/255A mutant (D1-LALA). 12CA5 HA antibodies were used forimmunoprecipitation of whole cell extracts prepared from these cells andco-immunoprecipitation of cyclin D1 (mutants) was examined by Westernblot analysis using anti-cyclin D1 antibody. The results showed thatwild-type cyclin D1, but not the leucine-to-alanine mutant D1-LALA,co-immunoprecipitates with SRC-1 and AIB-1, whereas binding of both wildtype cyclin D1 and D1 LALA mutant to p300 was hardly detectable.Precipitation with the anti-cyclin D1 antibody from the total lysate ofthe cells transfected with cyclin D1 and cyclin D1-LALA was similar.Binding of cyclin D1 and cyclin D1 L254/255A mutant to ER was theninvestigated. COS-7 cells were transfected with ER expression vector,cyclin D1 (mutant) and/or control plasmids. Monoclonal ER antibodieswere used for immunoprecipitation of ER of whole cell extracts preparedfrom these cells and co-immunoprecipitation of cyclin D1 (mutant) wasexamined by Western blot analysis using monoclonal cyclin D1 antibodies.The results show that co-immmunoprecipitation was similar for the (ERand cyclin D1) and (ER and cyclin D1-LALA) samples, showing that themutant was expressed equally and was unaffected in its ability to bindER. The activity of cyclin D1 and D1 L254/255A mutant in phosphorylationof pRb in Rb^(−/−) 3T3 cells was also investigated. Cells weretransfected with pRb expression vector, cyclin D1 (mutant) and/orcontrol plasmids and were maintained in low serum conditions. After 40hours, cells were lysed and proteins were separated by low percentagepolyacrylamide gel electrophoresis. Differentially phosphorylatedspecies of pRb were detected by Western blotting using the polyclonalpRb antibody (C15, Santa Cruz). Strong bands were seen in the Rb/cyclinD1 and Rb/cyclin D1-LALA bands but not in the control Rb band showingthat the mutant was unaffected in its ability to phosphorylate pRb incells that lack cyclin D1-associated kinase activity.

In summary, these three experiments show that the cyclin D1 mutantL254/255A, which failed to activate ER (FIG. 1B), did not interact withSRC family proteins, though this mutant was expressed equally and wasunaffected in its ability to bind ER and to phosphorylate pRb in cellsthat lack cyclin D1-associated kinase activity. These data suggest thatthe AF-2-like leucine-rich motif of cyclin D1 mediates binding toSRC-family proteins.

To test whether the interaction between cyclin D1 and SRC-1 is direct,we used bacterially expressed GST-SRC1 and E. coli-produced 6Xhistidine-tagged cyclin D1 in an in vitro protein binding assay. Asdescribed below, His-cyclin D1 strongly binds to GST-SRC1 protein, butnot to GST alone, indicating that this association is specific anddirect.

To ask which domain or motif of SRC-1 is involved in cyclin D1 binding,we generated a series a GST-SRC1 deletion mutants. The series of GSTfusion protein containing SRC-1 (GST-SRC1: 361-1441; GST-SRC1 361-782;GST-SRC1 361-568) or GST-p300(1-595) were tested for direct binding toHis-tagged cyclin D1 (His-D1); the GST-SRC1 derivatives are shown inFIG. 4A. The conserved LXXLL (SEQ ID NO:6) motif are boxed and the aminoacid boundaries are demonstrated. In the in vitro binding assay, theseries of GST-containing proteins were incubated with bacteriallyexpressed His-tagged cyclin D1 and immobilized on glutathione-agarose.Cyclin D1 binding was detected by Western blot analysis usinganti-cyclin D1 antibody. Binding was not detected to GST alone. Bindingwas detected to the GST-SRC1 361-1441, GST-SRC1 361-782 but not toGST-SRC1 361-568 or GST-p300 (1-595) proteins, showing that amino acids568 to 782 of SRC-1 are required for cyclin D1 binding. Interestingly,this region of SRC-1 harbors three LXXLL (SEQ ID NO:6) motifs, which areinvolved in binding to nuclear receptors (Heery et al., 1997; Le Douarinet al., 1995; Torchia et al., 1997).

To ask whether the LXXLL (SEQ ID NO:6) motifs of SRC-1 are involved incyclin D1 binding, a peptide competition experiment was performed.Peptides (0.3 μg and 3 μg) derived from the four LXXLL motifs of SRC1were used in a GST pull down assay using GST-SRC1 (361-1441) andHis-tagged cyclin D1 as described above. The sequence and the positionof the peptides in SRC-1 are shown in FIG. 4B. The results of the assayshowed that LXXLL (SEQ ID NO:6) peptides, but not a LXXAA mutantpeptide, interfered with binding of cyclin D1 to SRC-1. The reduction inbinding was greater with 3 μg than with 0.3 μg of each of the LXXLL (SEQID NO:6) peptides with no reduction shown with the LXXAA (SEQ IDNO:21)mutant peptide. Of the four SRC-1 LXXLL (SEQ ID NO:6) peptidestested, the P3 peptide, which corresponds to the third of the threecentrally located LXXLL motifs, was the best competitor. In anotherexperiment, the ability of peptides to compete with cyclin D1 forbinding to GST-SRC1 was investigated. Increasing amounts (100, 200 and500 nM) of AASKHKQLSELLRSG (SEQ ID NO:1) (LXXLL (SEQ ID NO:6)) andAASKHKQLSEAARSG (SEQ ID NO:23) (LXXAA) (SEQ ID NO:21) peptides were usedin a GST pull down assay using GST-SRC1 (361-1441) and His-taggedcycliin D1. Binding was reduced with increasing concentration of LXXLL(SEQ ID NO:6) peptides but not with increasing concentration of LXXAA(SEQ ID NO:21) peptides. These results indicate that SRC-1 uses theLXXLL (SEQ ID NO:6) motifs not only to interact with nuclear receptors,but also to bind cyclin D1. Since these LXXLL (SEQ ID NO:6) motifsinteract with the leucine-rich motifs on nuclear receptors (Heery etal., 1997; Torchia et al., 1997), these data are in good agreement withour experiments described above which suggested a major role for theleucine-rich AF-2-like motif of cyclin D1 in SRC binding. Since both thein vitro and the in vivo association experiments described above wereperformed in the absence of 17β-estradiol, these data also indicate thatthe cyclin D1/SRC1 interaction, in contrast to the ER/SRC-1 interaction,is hormone-independent.

Cyclin D1 Act as a Physical Bridge Between ER and SRC-1.

Cyclin D1 activates the estrogen receptor (ER) in a ligand-independentfashion through direct binding to ER (Neuman et al., 1997; Zwijsen etal., 1997). The present study demonstrates that cyclin D1, besides ER,also interacts directly with SRC-1 in vivo and in vitro. These datasuggest a model in which cyclin D1 can act as a bridging factor betweenER and SRC-1, allowing ligand-independent recruitment of coactivators toER in the presence of cyclin D1. To test this model directly, we usedbacterially expressed 6X histidine-tagged cyclin D1, E. coli-producedGST-SRC1 and baculovirus-produced ER in an in vitro GST pull down assay.Protein binding to GST-SRC1 was identified by Western blotting analysisusing monoclonal antibodies directed against cyclin D1 and ER. GSTprotein served as a control for binding specificity. In agreement withseveral earlier studies, we found that ER binds to GST-SRC1 in aligand-dependent manner in vitro (Cavailles et al., 1994) (FIG. 5A).Significantly, cyclin D1 could also recruit ER to GST-SRC1 in theabsence of ligand (FIG. 5A). These data indicate that cyclin D1 cancause ER activation by acting as a ligand-independent adapter moleculebetween ER and its coactivator SRC-1.

To test whether a ternary complex can also be formed when ER is bound toits cognate DNA binding site (the ERE), we performed a similar ternarycomplex assay with purified proteins as described above, with themodification that the ternary complex was pulled down with EREoligonucleotides coupled to paramagnetic beads. FIG. 5B shows that ERbinds to ERE in the presence of ligand (lanes 3-6), but also in theabsence of ligand when cyclin D1 is present (lanes 8 and 10). Thus,cyclin D1 allows ligand-independent DNA binding of ER to its ERE. Moreimportantly, this assay clearly shows that a ternary complex consistingof ER, cyclin D1 and ER can be formed on DNA both in the presence and inthe absence of 17β-estradiol (lanes 6 and 10). These data suggest thatcyclin D1 is a bridging factor between ER and SRC-1 also when ER isbound to DNA.

The Role of Cyclin D1 in ER Activation in Breast Cancer.

To get more insight in the role of cyclin D1 in ER transactivation inbreast cancers with elevated levels of cyclin D1, we have tested theeffect of the mutant cyclin D1 L254/255A on ER activation in twodifferent breast cancer cell lines. This cyclin D1 mutant does bind toER, but fails both to interact with SRC-1 as shown above andconsequently fails to activate ER (FIG. 1B). Therefore, this mutant canact as a dominant negative in cyclin D1-mediated ER transactivation asit binds to ER and fails to recruit coactivators. We tested the effectof this cyclin D1 mutant on ER activation in two breast cancer celllines, T47D and MCF-7, which contain endogenous wild type ER, but differin their cyclin D1 protein levels (FIG. 6). In T47D, which containrelative low levels of cyclin D1, co-expression of this cyclin D1 mutantresulted in a slight inhibition of ER activity. In contrast, in MCF-7,which contain relative high levels of cyclin D1 (FIG. 6), the cyclin D1mutant inhibited up to 50% of ER activity. Thus, this dominant negativecyclin D1 mutant preferentially interferes with ER activation in breastcancer cells that have high levels of cyclin D1 protein. These datastrongly support the notion that elevated cyclin D1 protein levels inbreast cancer contribute significantly to ER activation.

In parallel experiments, we have used the above dominant negative mutantof cyclin D1 to show that activation of the estrogen receptor complex byD1 stimulates the cell cycle. Cell cycle profiles were analysed usingfluuorescence activated cell sorting (FACS)(Allen, 1990; Baldetorp etal, 1998). Transient transfection of MCF-7 breast cancer cells with theL254/255A mutant of cyclin D1 causes an arrest in the cell cycle at theG1/S checkpoint (Table 2), thus confirming the role of the estrogenreceptor-cyclin D1 interaction in cell proliferation.

TABLE 2 Phase of cell cycle G0/G1 S G2/M No cyclin D1 73.4% 18.8% 7.8%Cyclin D1 76.5% 16% 7.6% Cyclin D1-LALA 81.9% 10.2% 7.9%

In summary, the invention indicates that cyclin D1 can act as a bridgingfactor between ER and steroid receptor coactivators (SRCs) which allowsthe formation of a transcriptionally active ternary complex in theabsence of ligand (FIG. 7). It is generally thought that coactivatorrecruitment by nuclear receptors results from a ligand-inducedconformational change in the AF-2 domain of the receptor (Brzozowski etal., 1997; Renaud et al., 1995). Our present data for the first timeshow an alternative route of coactivator recruitment to ER that can takeplace in the absence of ligand. As such, these data reveal a novelmechanism of ER activation and establish a direct role for cyclin D1 inregulation of transcription.

Our work has led to the identification of a novel functional domain inthe carboxyl terminus of cyclin D1 that mediates direct binding tosteroid receptor coactivators like SRC-1 and AIE-1. This leucine-richmotif of cyclin D1 is very similar in character to the ligand-regulatedsteroid receptor coactivator binding motif that is present in helix 12of ER and in many other nuclear receptors. Several lines of experimentalevidence indicate that the leucine-rich motif of cyclin D1 is requiredfor coactivator recruitment to ER and subsequent activation of ER.First, cyclin D1 interacts directly with SRC-1 both in vivo and in vitroand introduction-of two point mutations in this motif of cyclin D1abolishes SRC-1 interaction and prevents ER activation by cyclin D1.Second, cyclin D2 and D3 have only a partial conservation of theleucine-rich motif and are hardly active in ER activation (Neuman etal., 1997; Zwijsen et al., 1997). Third, a dominant negative mutant ofSRC-1 prevents cyclin D1 activation of ER, indicating that SRCs arerequired for cycdin D1-mediated activation of ER (FIG. 3). Fourth, in invitro binding studies cyclin D1 could recruit ER to SRC-1 in the absenceof ligand (FIG. 5). Together these data suggest a model in which cyclinD1 can recruit SRCs to ER which results in a transcriptionallyproductive interaction between ER and its coactivators.

The functional similarity between the leucine-rich motifs of cyclin D1and ER is also supported by structural analysis. Even though a crystalstructure of cyclin D1 is not available at present, the crystalstructures of cyclins A and H have been solved (Andersen et al., 1997;Jeffrey et al., 1995). Alignment of the sequence of cyclin D1 withcyclin A and comparison with the structures of cyclins A and H indicatesthat the leucine-rich motif in the carboxyl terminus of cyclin D1 alignsat the C-terminus of helix 5′ of cyclins A and H. Importantly, the PHDprogram indicates that this part of cyclin D1 has a more than 90%probability to be a-helical and is markedly amphipathic (Rost andSander, 1993). Since the leucine-rich coactivator binding motif of ER isalso an amphipathic helix, it is well-possible that the leucine-richmotifs of cyclin D1 and ER are capable of making similar proteininteractions with SRCs.

Consistent with the notion that cyclin D1 and ER have a similarcoactivator interaction surface, we found that binding of cyclin D1 toSRC-1 also requires the highly conserved LXXLL motifs in SRC-1. Thesemotifs were recently shown to mediate binding to the leucine-richcoactivator binding site in the amphipathic helix 12 of ER (Heery etal., 1997; Le Douarin et al., 1995; Torchia et al., 1997).Significantly, depending on the pattern of splicing, SRC-1 has three orfour LXXLL motifs, three of which are in close proximity (Kalkhoven etal., 1998). In principle, this could allow simultaneous interaction ofSRC-1 with the leucine-rich motifs of both ER and cyclin D1. Consistentwith this, we observed that a peptide that spans the third LXXLL motifof SRC-1 competed most efficiently the binding between cyclin D1 andSRC-1, whereas previous studies have indicated that the second LXXLLmotif of SRC-1 is the preferred site of interaction for ER (Heery etal., 1997; Kalkhoven et al., 1998). Based on these observations wepropose that in the absence of ligand, expression of cyclin D1 providesa single interaction site for coactivators on the cyclin D1/ER complexas both binding of cyclin D1 to ER and binding of cyclin D1 to SRC-1 isligand-independent. This provides a rationale for the ligand-independentactivation of ER in the presence of high levels of cyclin D1 (Zwijsen etal., 1997). After ligand binding of ER, the leucine-rich domain in AF-2is exposed which constitutes a second binding site for SRCs. Thepresence of two SRC-1 binding sites on the liganded cyclin D1/ER complexprovides a rationale for the observed synergism between estradiol andcyclin D1 in ER activation (Zwijsen et al., 1997, FIG. 7).

The model represented in FIG. 7 does not take into account that bindingof cyclin D1 to ER also allows ligand-independent DNA binding by ER invitro and in vivo (FIG. 5B and Zwijsen et al., 1997). Thus, cyclin D1can not only stimulate coactivator recruitment to ER but also act toenhance DNA binding of ER. Thus, the synergistic action between cyclinD1 and ligand in ER activation may also be due, in part, to synergisticinduction of ER DNA binding (FIG. 2 and Zwijsen et al., 1997).

The present study showed that cyclin D1 can bind to SRC-1 and AIB-1, butnot to p300. Thus, cyclin D1 can discriminate between the differentcoactivator families. Apparently, a LXXLL motif (present in both SRCsand p300) is required for cyclin D1 binding, but flanking regionscontribute to binding specificity (see also FIG. 4B). The finding thatcyclin D1 interacts with at least two members of the SRC family, SRC-1and AIB-1, would allow in principle for promiscuous activation ofsteroid receptors by cyclin D1. However, cyclin D1 does not activate theprogesterone receptor, nor a number of other steroid hormone receptors(Zwijsen et al., 1997) (R. M. L. Z. and R. B, unpublished data). It islikely that the ability of cyclin D1 to interact with ER directlycontributes to the specificity of nuclear receptor activation by cyclinD1.

Cyclin D1 is an important regulator of growth and differentiation ofbreast epithelium (Fantl et al., 1995; Musgrove et al., 1994; Sicinskiet al., 1995; van Diest et al., 1997; Wang et al., 1994; Zwijsen et al.,1996). Significantly, both the genes encoding cyclin D1 and the steroidreceptor coactivator AIB-1 are frequently amplified or overexpressed inbreast cancer (Anzick et al., 1997; Buckley et al., 1993; Gillett etal., 1994; Schuuring et al., 1992; van Diest et al., 1997). Since thepresent study indicates that both cyclin D1 and SRCs are components of amultimeric complex involved in ER-mediated transcription, it isconceivable that over-expression of limiting factors in this complexresults in deregulation of ER-mediated growth. In agreement with this,we found that a mutant of cyclin D1 that can bind to ER but fails torecruit coactivators acted as a dominant negative mutant for ERactivation primarily in breast cancer cells with elevated levels ofcyclin D1. Thus, cyclin D1 is likely to contribute significantly to ERactivation in breast cancers in which the protein is over-expressed. Animportant question that we wish to address next is how much of theoncogenic activity of cyclin D1 in breast cancer is mediated through the“classical” cdk4 route and how much through ER activation. Theavailability of specific mutants of cyclin D1 in which these activitiescan be separated should allow us to assess the contribution of each ofthese two activities of cyclin D1 to mammary carcinogenesis separately.

EXPERIMENTAL PROCEDURES

Cell Culture and Transient Transfection Assays.

COS-7 cells and U2-OS cells were maintained in DMEM supplemented with10% fetal bovine serum. Twenty-four hours before transfection, cellswere maintained in DMEM without phenol red containing 5%charcoal-treated FBS. Cells were transfected with 3 μgERE-TATA-luciferase expression vector, 500 ng β-galactosidase expressionvector (internal control);, 200 ng ER expression plasmid and 2.5 μgcyclin D1, coactivators and/or empty vectors as indicated. After sixteenhours, cells were rinsed in PBS and re-fed with fresh medium and ligand(10 nM 17β-estradiol) or vehicle was added. One day later, cells wereharvested and assayed for luciferase and β-galactosidase activities.β-galactosidase activity was used to correct for differences intransfection efficiency.

Immunoprecipitation and Western Blotting.

Cells were lysed in ELB containing 250 mM NaCl, 0.1% NP-40, 50 mM HEPESpH 7.0, 5 mM EDTA and protease inhibitors. The cell lysate waspre-cleared three times with 5 μl of normal mouse serum coupled toprotein A-Sepharose beads. For immunoprecipitations, the supernatant wasincubated with 100 μl of 12CA5 hybridoma supernatant or 10 μl monoclonalantibodyto the estrogenreceptor (TE111.5D11, Neomarkers), which wascoupled to protein A sepharose beads at 4° C. After 1 hour, beads werewashed in ELE buffer and boiled in Laemmli-buffer. Samples wereseparated on a 10% SDS/polyacrylamide gel and transferred tonitrocellulose. After blocking with PBS containing 5% milk and 0.1%Tween-20, proteins were detected with monoclonal antibodies directedagainst cyclin D1 (DCS-6, Neomarkers) and peroxidase-conjugated goatanti-mouse IgG. The blots were washed in PBS containing 0.1% Tween-20and developed by enhanced chemiluminescence (ECL) reactions (Amersham).

DNA Binding Assay.

For the DNA binding assay we used DNA affinity beads coated withstreptavidin (Dynal A/S) and (5-biotin-labeled) DNA oligonucleotidescontaining a binding sequence for ER as described before (Zwijsen etal., 1997). The complementary DNA strands were annealed in TE buffercontaining 100 mM KCl at 75 ûC for 10 min. followed by cooling to roomtemperature over a period of 2 h. Dynabeads were mixed with biotinylatedoligonucleotides in TE buffer containing 1 M NaCl for 15 min., washedand incubated with cell extract in 8 mM Tris-phosphate pH 7.4, 0.12 KCl,8% glycerol, 4 mM DTT and 0.5% CHAPS for 1 h at 4° C. Subsequently,beads were washed in 20 mM Hepes pH 7.7, 50 mM KCl, 20% glycerol and0.1% NP-40. The beads were boiled in Laemlli buffer and the proteinswere separated on 10% PAGE and identified by Western blotting.

GST Pull Down and Peptide Competition Assay.

GST protein, GST-SRC1 fusion protein and His-tagged cyclin D1 proteinwere purified as described previously (Zwijsen et al., 1997). Bindingbetween 500 ng GST-SRC1 and 100 ng His-D1 was performed in bindingbuffer (50 mM NaCl, 50 mM HEPES-KOH pH 7.6, 0.1 mM 0.1% (w/v) NP40, 0.1mM PMSF and 0.5% charcoal-stripped serum) bound to glutathione-sepharosefor 1 hour at 4° C. The beads were washed three times and bound proteinswere eluted by boiling for 10 minutes in sample buffer and separated on10% SDS-PAGE. The binding of His-cyclin D1 to GST-SRC1 was detected byWestern blot analysis using monoclonal antibodies directed againstcyclin D1 (DCS-6, Neomarkers). For testing a ternary complex, abaculovirus-produced ER (750 ng, Pan Vera) was added to GST-SRC1 (500ng) and His-D1 (100 ng) in the presence or absence of 1 μM 17β-estradiolusing similar conditions as described above. In Western blot analysismonoclonal antibodies directed against cyclin D1 (DCS-6, Neomarkers) andER (LH2, Novocastra) were used.

For the peptide inhibition assay using the peptides shown in FIG. 4(B),150 ng GST/GST-SRC1 and 50 ng His-tagged cyclin D1 were used. Peptideswere pre-incubated with target protein for 40 minutes at roomtemperature, prior to addition of the bait. A mixture of GST-fusionpeptides, His-tagged cyclin D1 and peptides was incubated for anadditional 20 min. at room temperature. The amount of peptides added incompetition studies were 0.3 μg and 3 μg. For the peptide inhibitionassay using the peptides AASKHKQLSELLRSG (SEQ ID NO:1)andAASKHKQLSEAARSG (SEQ ID NO:23) derived from p300 amino acid 74-88, thepeptides were pre-incubated with target protein for 20 minutes at roomtemperature, prior to addition of the bait. The peptides added incompetition studies were 100 nM, 200 nM and 500 nM.

The Following Literature Cited Herein is Incorporated by Reference.

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1. An isolated peptide of formula:Z¹-LXXLL-Z² (SEQ ID NO:9) where at least one X is a charged amino acid;Z¹ comprises from 3 to 7 amino acids and contains at least one residueselected from the group consisting of K and H; and Z² is a C-terminal orsequence of from 1 to 8 amino acids.
 2. A isolated peptide according toclaim 1, wherein Z¹ and/or Z² represent a sequence found immediatelyN-terminal and/or C-terminal respectively to a LXXLL (SEQ ID NO:6) motifin an SRC or other nuclear receptor interacting protein.
 3. A isolatedpeptide consisting of a formula selected from the group. A_A S K H K Q LS E L L R S G (SEQ ID NO:1)         S H K L V Q L L T T T_A E Q (SEQ IDNO:2)     E R H K I L H R L L Q E G S (SEQ ID NO:3)       K D H Q L L RL L D K D E (SEQ ID NO:4); and P Q A Q Q K S L Q Q L L T (SEQ ID NO:5).


4. A isolated peptide whose sequence comprises a peptide as defined inclaim 1 or 3 joined to a membrane translocation sequence.
 5. Acomposition comprising a isolated peptide according to claim 1 or 3together with a diluent or carrier.